The invention relates to products and methods for medical treatment of Gaucher disease and, in particular, nucleic acid molecules, polypeptides and vectors for polypeptide or gene therapy treatment.
Gaucher disease is a lysosomal storage disease caused by the deficiency of functional glucocerebrosidase (Gcc) enzyme. Gcc is present in all cell types. The defective enzyme cannot break down a fatty substance, glucocerebroside, which is an important component of cell membranes. The fat accumulates in macrophages (which are known as the xe2x80x9cGaucher cellsxe2x80x9d). The fat-laden macrophages are found typically in the liver, spleen, bone marrow and lungs. The amount of the enzyme deficiency varies from person to person as do the symptoms. Some patients may show no clinical symptoms, while others may die from the disease. The symptoms of the disease and mutant forms of Gcc that cause Gaucher disease are described, for example, in U.S. Pat. No. 5,266,459 (Beutler) and U.S. Pat. No. 5,234,811 (Beutler and Sorge).
There are therapies for Gaucher disease. Ceredase is a form of the Gcc enzyme from placenta that is able to metabolize the fat in Gaucher cells. The enzyme restores normal function to a Gaucher cell. The amount of enzyme used in treatment varies. As much as 30-60 units per kilogram of bodyweight (U/kg/bw) may be given every other week. Positive results have been reported with 2.3 U/kg/bw given three times a week. Lower doses, such as 1-5 U/kg/bw twice weekly, have also been used with success, but this is less frequent. The intarcellular half life of the enzyme is up to 60 hours. A large number of placentas are needed to make sufficient Ceredase, so this form of therapy is very expensive. It has been almost completely replaced by treatment with a recombinant form of the enzyme, Cerezyme but this therapy is also expensive. Cerezyme is dispensed as a powder whereas Ceredase comes as a liquid. Sterile water must be added to the Cerezyme bottle to dissolve the powder. The shelf life of the drugs is short ( less than 3 months), and splitting doses is cumbersome and wasteful. Allergic reactions to Ceredase are common, but rarely life-threatening. Adverse reactions to Cerezyme appear to be less common, but experience with the drug is still very limited.
Gcc has been structurally modified in order to obtain improved pharmacokinetics over naturally occurring Gcc (which is derived from placenta). These modifications include amino acid modifications as well as carbohydrate changes. For example, U.S. Pat. No. 5,549,892 discloses a recombinant polypeptide that differs from naturally occurring Gcc by the presence of histidine in place of arginine at position 495. In another embodiment, the carbohydrate remodeled recombinant Gcc has increased fucose and N-acetyl glucosamine residues compared to remodeled naturally occurring Gcc. The increased pharmacokinetics of these compounds provides a therapeutic effect at doses that are lower than those required using remodeled, naturally occurring Gcc. However, this Gcc remains expensive to provide. Furthermore, improved pharmacokinetics does not necessarily compensate for inadequate bioavailability of Gcc.
Gene therapy has been administered to Gaucher patients. All experiments carried out to date have been undertaken using ex vivo, retrovirus-mediated transfection, which requires sophisticated laboratory facilities and is very expensive. Although transgene expression could be demonstrated in mice undergoing this procedure, experiments in humans have been disappointing. No clinically significant Gcc gene expression has been reported in humans undergoing retrovirus-mediated transfection with existing Gcc gene preparations. One problem of gene therapy is in reproducibly obtaining high-level, tissue-specific and enduring expression from genes transferred into cells. Currently, there is no suitable gene therapy vector that expresses at a high level for Gaucher disease gene therapy.
The invention includes a modified Gcc cDNA insert that can be inserted into any mammalian expression vector for use in the medical treatment of Gaucher disease. In a preferred embodiment, the modified cDNA was inserted into a vector named pINEX2.0 which was then used to transfect mammalian cells. When pINEX2.0 containing the unmodified Gcc cDNA coding sequence, pINEX5xe2x80x2GCC3xe2x80x2, was transfected into cells, their RNA purified from cell lysates and subjected to reverse transcription followed by the polymerase chain reaction (RT-PCR), two distinct major bands were observed after agarose gel electrophoresis (FIG. 1). Isolation, purification and sequencing of the RT-PCR products identified a major aberrantly spliced mRNA species which encodes only a 19 amino acid peptide before encountering a STOP codon. Surprisingly, this aberrant splicing event occurred completely within the Gcc cDNA coding sequence (FIG. 2), i.e. no vector sequences were involved. Site directed mutagenesis was performed to modify the nucleotide sequence in the region of aberrant mRNA splicing without affecting polypeptide coding (FIG. 3; SEQ ID NOS.: 2, 4, and 6). Modifications were aimed at disrupting the known consensus sequences for RNA-splicing (Krawczak et al. 1992). The effectiveness of these modifications were tested by transient transfection into CHO cells, followed by our human-specific immunoprecipitation assay for Gcc. Data (n=18) indicate a 5xc2x11 (Std. Error)-fold increase in Gcc activity was achieved when the modified replaced the unmodified insert in the pINEX2.0 expression vector.
The invention relates to an isolated Gcc DNA molecule, wherein the DNA molecule has a modification in at least one nucleotide that disrupts a splicing consensus sequence and prevents splicing of mRNA produced from the DNA molecule, while preserving the ability of the DNA to express active Gcc. The modification impairs a consensus nucleotide sequence needed to induce splicing. The DNA molecule is preferably modified at two cryptic splice sites. The DNA preferably includes a mutation in the 3xe2x80x2 junction site. In one embodiment, the mutation is as shown in the 3xe2x80x2 junction site (SEQ ID NO.: 18) in Table 1, or a functionally equivalent mutation. In another embodiment, the DNA molecule includes a mutation in the 5xe2x80x2 splice junction site. The mutation is preferably as shown in the 5xe2x80x2 junction site (SEQ ID NO.: 19) in Table 1, or a functionally equivalent mutation.
The DNA molecule preferably includes all or part of the nucleotide sequence shown in FIG. 5 (SEQ ID NO.: 13).
Another aspect of the invention relates to a vector including a DNA molecule of the invention. The vector preferably includes a promoter that is functional in a mammalian cell.
The invention also includes mRNA produced from the DNA molecule or vector of the invention.
Another aspect of the invention relates to a method of medical treatment of Gaucher disease in a mammal, including administering to the mammal an effective amount of a nucleic acid molecule of the invention or a vector of the invention and expressing an effective amount of the polypeptide encoded by the nucleic acid molecule for alleviating clinical symptoms of Gaucher disease.
The invention includes a host cell, or progeny thereof, including a nucleic acid molecule of the invention. The host cell is preferably selected from the group consisting of a mammalian cell, a human cell and a Chinese Hamster Ovary cell. The invention also includes a method for producing a recombinant host cell capable of expressing a Gcc nucleic acid molecule, the method including introducing into the host cell a vector of the invention. The invention also includes a method for expressing a Gcc polypeptide in a host cell including culturing the host cell under conditions suitable for DNA molecule expression. Another aspect of the invention relates to a method for producing a transgenic cell that expresses elevated levels of Gcc polypeptide relative to a non-transgenic cell, including transforming a cell with a vector of the invention.
The invention includes an isolated polypeptide encoded by and/or produced from a nucleic acid molecule of the invention, or a vector of the invention.
The invention includes a method of producing a genetically transformed cell which expresses or overexpresses a Gcc polypeptide, including: a) preparing a Gcc nucleic acid molecule according to any of claims 1-18; b) inserting the nucleic acid molecule in a vector so that the nucleic acid molecule is operably linked to a promoter; c) inserting the vector into a cell. The invention includes a transgenic cell produced according to the method of the invention.
The invention also includes a pharmaceutical composition, including a carrier and (i) a nucleic acid molecule of the invention (ii) a vector of the invention or (iii) Gcc polypeptide produced from (i) or (ii), in an effective amount for reducing clinical symptoms of Gaucher disease. The carrier preferably carrier includes a liposome.